Relevance: Herpes simplex virus (HSV-1) continues to be the cause of widespread human illness despite the availabiltiy of a reasonably effective therapeutic, acyclovir, and related compounds. Diseases caused by HSV-1 include disseminated illness in the newborn, cold sores, genital lesions, non-epidemic encephalitis in adults, a stromal keratitisand retinits. The goal of the proposed project is to study assembly of the virus capsid with the aim of identifying novel targets against which small molecule HSV-1 inhibitors might be directed. Project Summary: Like all herpesviruses, HSV-1 consists of an icosahedral capsid surrounded by a membrane envelope. The capsid, which contains the virus DMA, is assembled in the infected cell nucleus. A DNA-free capsid shell is first formed and later packaged with DNA. Capsids are assembled from a major structural protein (UL19), three other structural polypeptides and a scaffolding protein. Capsids must be formed in such a way that they are able to release the encapsidated DNA as a new cycle of infection is nitiated. Individual steps in capsid assembly and DNA uncoating are regarded as attractive targets for novel therapeutics because they are required for HSV-1 replication and because virus-encoded proteins are the primary components involved. In their basic features, the steps of capsid assembly and DNA egress in HSV- 1 are expected to be the same as those for other herpesviruses. Thus potential drug targets identified in HSV-1 should be able to be exploited for the design of therapeutics effective against other herpesviruses including human cytomegalovirus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. The major new initiative proposed here is an analysis of how DNA is released from the capsid to initiate a new infection. We propose to test the idea that proteolytic digestion of one or more capsid proteins in functionally involved in promoting DNA egress (aims 1 and 2). Other proposed studies make use of a cell-free capsid assembly system to examine the way the portal becomes incorporated into the nascent capsid. We propose to test the hypothesis that assemlby involves a filamentous aggregate of the scaffolding protein bound to the portal and major capsid proteins (aim 3). This is proposed to condense in a stepwise fashion to create the procapsid. Studies will also be performed to identify how components of the DNA packaging machinery are assembled on the capsid surface prior to their functining in DNA translocation (aim 4).